RNAi targeting of rootworm Troponin I transcripts confers root protection in maize

Abstract

Western corn rootworm, Diabrotica virgifera virgifera, is the major agronomically important pest of maize in the US Corn Belt. To augment the repertoire of the available dsRNA-based traits that control rootworm, we explored a potentially haplolethal gene target, wings up A (wupA), which encodes Troponin I. Troponin I, a component of the Troponin-Tropomyosin complex, is an inhibitory protein involved in muscle contraction. In situ hybridization showed that feeding on wupA-targeted dsRNAs caused systemic transcript knockdown in D. v. virgifera larvae. The knockdown of wupA transcript, and by extension Troponin I protein, led to deterioration of the striated banding pattern in larval body muscle and decreased muscle integrity. Additionally, the loss of function of the circular muscles surrounding the alimentary system led to significant accumulation of food material in the hind gut, which is consistent with a loss of peristaltic motion of the alimentary canal. In this study, we demonstrate that wupA dsRNA is lethal in D. v. virgifera larvae when fed via artificial diet, with growth inhibition of up to 50% within two days of application. Further, wupA hairpins can be stably expressed and detected in maize. Maize expressing wupA hairpins exhibit robust root protection in greenhouse bioassays, with several maize transgene integration events showing root protection equivalent to commercial insecticidal protein-expressing maize.

Delivery of gene‐specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, Myllocerus undecimpustulatus undatus Marshall

“…Transgenic plants engineered to express specific dsRNA molecules targeting essential genes have proven to be an efficient strategy to control various insect pests, [45][46][47][48] therefore this approach for dsRNA delivery could be also tested against SLW. In addition to transgenic plants, alternative systems to deliver dsRNA that are possible under field conditions, such as soil drench, stem and trunk injection, foliar application, and symbiotic bacteria and viruses, have been proposed.…”

Control of western corn rootwormviaRNAi traits in maize: lethal and sublethal effects ofSec23dsRNA

“…All Sec23 dsRNAs were amplified from WCR cDNA, while GFP was amplified from the pIZT/V5‐His expression vector (Invitrogen, Carlsbad, CA, USA). A previously‐described YFP dsRNA was used . All PCR amplification products were sequenced to confirm the identity and specificity of the sequence before dsRNA synthesis, which was performed using the MEGAscript high‐yield Transcription Kit (Applied Biosystems Inc., Foster City, CA, USA) as previously described .…”

“…Construct design and development of transgenic plants were performed as described by Fishilevich et al . Briefly, standard cloning methods were used to construct binary transformation plasmids based on pTI15955 from Agrobacterium .…”

“…A previously-described YFP dsRNA was used. 24 All PCR amplification products were sequenced to confirm the identity and specificity of the sequence before dsRNA synthesis, which was performed using the MEGAscript high-yield Transcription Kit (Applied Biosystems Inc., Foster City, CA, USA) as previously described. 23,30 Synthesized dsRNAs were purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions, quantified using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA), and examined by agarose gel electrophoresis to determine their purity and integrity.…”

“…17 While the sensitivity of insects to dsRNA varies greatly among insect orders, [18][19][20] rootworms are very sensitive to dsRNA applied to artificial diet 17,[21][22][23][24] or transgenically expressed in maize plants. 18,[23][24][25][26] Target sequences that have demonstrated plant protection against WCR include the vacuolar proton pump, V-ATPase A, 17 the Snf7/Vps32 subunit of the ESCRT-III (endosomal sorting complex required for transport III), 27 septate junction proteins snakeskin (dvssj1) and mesh (dvssj2), 26 Troponin I, 24 SNARE-binding protein Ras opposite/Sec1, RNA polymerase II subunit RpII140, and FACT complex proteins dre4/spt16. 23 The most common method to initially assess the efficacy of RNAi targets is by applying dsRNA to artificial diet to determine if the insect is susceptible to orally administered dsRNA and the efficacy of the selected target gene.…”